Pyruvate Loading Research Articles

Metformin acts as a dual glucose regulator in mouse brain.

Aims: Metformin improves glucose regulation through various mechanisms in the periphery. Our previous study revealed that oral intake of metformin activates several brain regions, including the hypothalamus, and directly activates hypothalamic S6 kinase in mice. In this study, we aimed to identify the direct effects of metformin on glucose regulation in the brain. Materials and methods: We investigated the role of metformin in peripheral glucose regulation by directly administering metformin intracerebroventricularly in mice. The effect of centrally administered metformin (central metformin) on peripheral glucose regulation was evaluated by oral or intraperitoneal glucose, insulin, and pyruvate tolerance tests. Hepatic gluconeogenesis and gastric emptying were assessed to elucidate the underlying mechanisms. Liver-specific and systemic sympathetic denervation were performed. Results: Central metformin improved the glycemic response to oral glucose load in mice compared to that in the control group, and worsened the response to intraperitoneal glucose load, indicating its dual role in peripheral glucose regulation. It lowered the ability of insulin to decrease serum glucose levels and worsened the glycemic response to pyruvate load relative to the control group. Furthermore, it increased the expression of hepatic G6pc and decreased the phosphorylation of STAT3, suggesting that central metformin increased hepatic glucose production. The effect was mediated by sympathetic nervous system activation. In contrast, it induced a significant delay in gastric emptying in mice, suggesting its potent role in suppressing intestinal glucose absorption. Conclusion: Central metformin improves glucose tolerance by delaying gastric emptying through the brain-gut axis, but at the same time worsens it by increasing hepatic glucose production via the brain-liver axis. However, with its ordinary intake, central metformin may effectively enhance its glucose-lowering effect through the brain-gut axis, which could surpass its effect on glucose regulation via the brain-liver axis.

Regulation of basal expression of hepatic PEPCK and G6Pase by AKT2.

Hepatic glucose metabolism signaling downstream of insulin can diverge to multiple pathways including AKT. Genetic studies suggest that AKT is necessary for insulin to suppress gluconeogenesis. To specifically address the role of AKT2, the dominant liver isoform of AKT in the regulation of gluconeogenesis genes, we generated hepatocytes lacking AKT2 (Akt2-/-). We found that, in the absence of insulin signal, AKT2 is required for maintaining the basal level expression of phosphoenolpyruvate carboxyl kinase (PEPCK) and to a lesser extent G6Pase, two key rate-limiting enzymes for gluconeogenesis that support glucose excursion due to pyruvate loading. We further showed that this function of AKT2 is mediated by the phosphorylation of cyclic AMP response element binding (CREB). Phosphorylation of CREB by AKT2 is needed for CREB to induce the expression of PEPCK and likely represents a priming event for unstimulated cells to poise to receive glucagon and other signals. The inhibition of gluconeogenesis by insulin is also dependent on the reduced FOXO1 transcriptional activity at the promoter of PEPCK. When insulin signal is absent, this activity appears to be inhibited by AKT2 in manner that is independent of its phosphorylation by AKT. Together, this action of AKT2 on FOXO1 and CREB to maintain basal gluconeogenesis activity may provide fine-tuning for insulin and glucocorticoid/glucagon to regulate gluconeogenesis in a timely manner to meet metabolic needs.

Prolonged fasting elicits increased hepatic triglyceride accumulation in rats born to dexamethasone-treated mothers

We investigated the effect of dexamethasone during the last week of pregnancy on glucose and lipid metabolism in male offspring. Twelve-week old offspring were evaluated after fasting for 12-hours (physiological) and 60-hours (prolonged). Physiological fasting resulted in glucose intolerance, decreased glucose clearance after pyruvate load and increased PEPCK expression in rats born to dexamethasone-treated mothers (DEX). Prolonged fasting resulted in increased glucose tolerance and increased glucose clearance after pyruvate load in DEX. These modulations were accompanied by accumulation of hepatic triglycerides (TG). Sixty-hour fasted DEX also showed increased citrate synthase (CS) activity, ATP citrate lyase (ACLY) content, and pyruvate kinase 2 (pkm2), glucose transporter 1 (slc2a1) and lactate dehydrogenase-a (ldha) expressions. Hepatic AKT2 was increased in 60-hour fasted DEX, in parallel with reduced miRNAs targeting the AKT2 gene. Altogether, we show that metabolic programming by prenatal dexamethasone is characterized by an unexpected hepatic TG accumulation during prolonged fasting. The underlying mechanism may depend on increased hepatic glycolytic flux due to increased pkm2 expression and consequent conversion of pyruvate to non-esterified fatty acid synthesis due to increased CS activity and ACLY levels. Upregulation of AKT2 due to reduced miRNAs may serve as a permanent mechanism leading to increased pkm2 expression.

A Liver-Specific Defect of Acyl-CoA Degradation Produces Hyperammonemia, Hypoglycemia and a Distinct Hepatic Acyl-CoA Pattern

Most conditions detected by expanded newborn screening result from deficiency of one of the enzymes that degrade acyl-coenzyme A (CoA) esters in mitochondria. The role of acyl-CoAs in the pathophysiology of these disorders is poorly understood, in part because CoA esters are intracellular and samples are not generally available from human patients. We created a mouse model of one such condition, deficiency of 3-hydroxy-3-methylglutaryl-CoA lyase (HL), in liver (HLLKO mice). HL catalyses a reaction of ketone body synthesis and of leucine degradation. Chronic HL deficiency and acute crises each produced distinct abnormal liver acyl-CoA patterns, which would not be predictable from levels of urine organic acids and plasma acylcarnitines. In HLLKO hepatocytes, ketogenesis was undetectable. Carboxylation of [2-14C] pyruvate diminished following incubation of HLLKO hepatocytes with the leucine metabolite 2-ketoisocaproate (KIC). HLLKO mice also had suppression of the normal hyperglycemic response to a systemic pyruvate load, a measure of gluconeogenesis. Hyperammonemia and hypoglycemia, cardinal features of many inborn errors of acyl-CoA metabolism, occurred spontaneously in some HLLKO mice and were inducible by administering KIC. KIC loading also increased levels of several leucine-related acyl-CoAs and reduced acetyl-CoA levels. Ultrastructurally, hepatocyte mitochondria of KIC-treated HLLKO mice show marked swelling. KIC-induced hyperammonemia improved following administration of carglumate (N-carbamyl-L-glutamic acid), which substitutes for the product of an acetyl-CoA-dependent reaction essential for urea cycle function, demonstrating an acyl-CoA-related mechanism for this complication.

Melatonin acts through MT1/MT2 receptors to activate hypothalamic Akt and suppress hepatic gluconeogenesis in rats

Melatonin can contribute to glucose homeostasis either by decreasing gluconeogenesis or by counteracting insulin resistance in distinct models of obesity. However, the precise mechanism through which melatonin controls glucose homeostasis is not completely understood. Male Wistar rats were administered an intracerebroventricular (icv) injection of melatonin and one of following: an icv injection of a phosphatidylinositol 3-kinase (PI3K) inhibitor, an icv injection of a melatonin receptor (MT) antagonist, or an intraperitoneal (ip) injection of a muscarinic receptor antagonist. Anesthetized rats were subjected to pyruvate tolerance test to estimate in vivo glucose clearance after pyruvate load and in situ liver perfusion to assess hepatic gluconeogenesis. The hypothalamus was removed to determine Akt phosphorylation. Melatonin injections in the central nervous system suppressed hepatic gluconeogenesis and increased hypothalamic Akt phosphorylation. These effects of melatonin were suppressed either by icv injections of PI3K inhibitors and MT antagonists and by ip injection of a muscarinic receptor antagonist. We conclude that melatonin activates hypothalamus-liver communication that may contribute to circadian adjustments of gluconeogenesis. These data further suggest a physiopathological relationship between the circadian disruptions in metabolism and reduced levels of melatonin found in type 2 diabetes patients.

Association of exercise-induced hyperinsulinaemic hypoglycaemia with MCT1-expressing insulinoma

Exercise-induced hyperinsulinism (EIHI) is a hypoglycaemic disorder characterised by inappropriate insulin secretion following anaerobic exercise or pyruvate load. Activating promoter mutations in the MCT1 gene (also known as SCLA16A1), coding for monocarboxylate transporter 1 (MCT1), were shown to associate with EIHI. Recently, transgenic Mct1 expression in pancreatic beta cells was shown to introduce EIHI symptoms in mice. To date, MCT1 has not been demonstrated in insulin-producing cells from an EIHI patient. In vivo insulin secretion was studied during an exercise test before and after the resection of an insulinoma. The presence of MCT1 was analysed using immunohistochemistry followed by laser scanning microscopy, western blot analysis and real-time RT-PCR of MCT1. The presence of MCT1 protein was analysed in four additional insulinoma patients. Clinical testing revealed massive insulin secretion induced by anaerobic exercise preoperatively, but not postoperatively. MCT1 protein was not detected in the patient's normal islets. In contrast, immunoreactivity was clearly observed in the insulinoma tissue. Western blot analysis and real-time RT-PCR showed a four- to fivefold increase in MCT1 in the insulinoma tissue of the EIHI patient compared with human pancreatic islets. MCT1 protein was detected in three of four additional insulinomas. We show for the first time that an MCT1-expressing insulinoma was associated with EIHI and that MCT1 might be present in most insulinomas. Our data suggest that MCT1 expression in human insulin-producing cells can lead to EIHI and warrant further studies on the role of MCT1 in human insulinoma patients.

Primary metabolism in the new human cell line AGE1.HN at various substrate levels: increased metabolic efficiency and α1-antitrypsin production at reduced pyruvate load

Metabolic responses of the new neuronal human cell line AGE1.HN to various substrate levels were analyzed in this study showing that reduced substrate and especially pyruvate load improves metabolic efficiency, leading to improved growth and α(1)-antitrypsin (A1AT) production. The adaptation of the metabolism to different pyruvate and glutamine concentrations was analyzed in detail using a full factorial design. The most important finding was an increasingly inefficient use of substrates as well as the reduction of cell proliferation with increasing pyruvate concentrations in the medium. Cultivations with different feeding profiles showed that the highest viable cell density and A1AT concentration (167% of batch) was reached in the culture with the lowest glucose level and without pyruvate feeding. Analysis of metabolic fluxes in the differently fed cultures revealed a more efficient metabolic phenotype in the cultures without pyruvate feeding. The measured in vitro enzyme activities of the selected enzymes involved in pyruvate metabolism were lower in AGE1.HN compared with CHO cells, which might explain the higher sensitivity and different adaptation of AGE1.HN to increased pyruvate concentrations. The results indicate on the one hand that increasing the connectivity between glycolysis and the TCA cycle might improve substrate use and, finally, the production of A1AT. On the other hand, a better balanced substrate uptake promises a reduction of energy spilling which is increased with increasing substrate levels in this cell line. Overall, the results of this study provide important insights into the regulation of primary metabolism and into the adaptation of AGE1.HN to different substrate levels, providing guidance for further optimization of production cell lines and applied process conditions.

Physical Exercise–Induced Hypoglycemia Caused by Failed Silencing of Monocarboxylate Transporter 1 in Pancreatic β Cells

Exercise-induced hyperinsulinism (EIHI) is a dominantly inherited hypoglycemic disorder characterized by inappropriate insulin secretion during anaerobic exercise or on pyruvate load. We aimed to identify the molecular basis of this novel disorder of beta -cell regulation. EIHI mapped to chromosome 1 (LOD score 3.6) in a genome scan performed for two families with 10 EIHI-affected patients. Mutational analysis of the promoter of the SLC16A1 gene, which encodes monocarboxylate transporter 1 (MCT1), located under the linkage peak, revealed changes in all 13 identified patients with EIHI. Patient fibroblasts displayed abnormally high SLC16A1 transcript levels, although monocarboxylate transport activities were not changed in these cells, reflecting additional posttranscriptional control of MCT1 levels in extrapancreatic tissues. By contrast, when examined in beta cells, either of two SLC16A1 mutations identified in separate pedigrees resulted in increased protein binding to the corresponding promoter elements and marked (3- or 10-fold) transcriptional stimulation of SLC16A1 promoter-reporter constructs. These studies show that promoter-activating mutations in EIHI induce SLC16A1 expression in beta cells, where this gene is not usually transcribed, permitting pyruvate uptake and pyruvate-stimulated insulin release despite ensuing hypoglycemia. These findings describe a novel disease mechanism based on the failure of cell-specific transcriptional silencing of a gene that is highly expressed in other tissues.

In vivo functional investigations of lactic acid in patients with respiratory chain disorders

OBJECTIVETo assess the prevalence of in vivo detectable abnormalities of lactate metabolism in mitochondrial disorders.DESIGNRetrospective study in a metabolic investigation unit.PATIENTS28 patients with a respiratory chain disorder identified from biochemical...

A mitochondrial encephalomyopathy with a partial cytochrome c oxidase deficiency of muscle.

A 16 year old girl showed delayed psychomotor development. In infancy, exercise intolerance, cerebellar signs, deteriorated with increasing intercurrent infections, and disturbances of breathing and cardiac rhythm became manifest. From the age of 7 years there was chronic progressive psychomotor deterioration, with hypotonia, a bilateral pyramidal and cerebellar syndrome, and mild epilepsy. CSF pyruvate and lactate levels were elevated, and lactate content was elevated in the urine. There was an abnormally high rise of lactate levels on moderate exercise and an abnormal response to pyruvate loading. Quadriceps muscle biopsies obtained at age 10 and 16 years showed ragged-red fibres, and a decreased cytochrome c oxidase activity and cytochrome aa3 content. Cytochrome c oxidase activity in fibroblasts was normal. Clinical signs and symptoms in association with a disturbance of mitochondrial energy metabolism led us to diagnosis of probable Leigh syndrome.

Intravenous pyruvate loading test in Leigh syndrome

Diagnosis of defective pyruvate metabolism can present difficulties in clinical practice. In search of a diagnostic procedure that can give a clear indication of a disturbance of pyruvate metabolism, we have developed an intravenous pyruvate loading test. The loading test was applied to 9 patients with Leigh syndrome. Results and characteristics are described. The test proved to be a sensitive procedure to detect disturbances in pyruvate oxidation. The intravenous pyruvate loading test can be a useful tool in the diagnosis of mitochondrial (encephalo)myopathies.

Friedreich's ataxia

Nine patients with typical Friedreich's ataxia had normal levels of pyruvate and lactate in blood, urine, and CSF. Glucose, pyruvate, and lactate levels were also within the reference ranges after oral glucose loading. After intravenous pyruvate loading, the relative increase in blood pyruvate and lactate levels was significantly higher in Friedreich's ataxia patients than in controls. Significant differences between the two groups were also observed in lactate-pyruvate ratios at various times after infusion. The pyruvate loading test can be useful in demonstrating a defect in pyruvate metabolism in Friedreich's ataxia patients.

Effects of endotoxin on gluconeogenesis, glycogen synthesis, and liver glycogen synthase in mice.

This study was undertaken to characterize the nature of carbohydrate loss due to endotoxin poisoning in mice and to elucidate mechanisms responsible for the changes. Female ICR mice, fasted overnight, were injected intraperitoneally with a mean lethal dose of endotoxin extracted from Salmonella typhimurium strain SR-11. Liver glycogen levels, alanine-U-(14)C and pyruvate-2-(14)C incorporation into blood glucose and liver glycogen, glucose-U-(14)C incorporation into liver glycogen, and liver glycogen synthase activities were measured at intervals after treatment. Liver glycogen in fasted mice given endotoxin was diminished significantly as early as 1 h after treatment. Liver glycogen synthase was significantly decreased in poisoned mice at 17 h. The use of actinomycin D showed that the induction of this enzyme due to fasting or hydrocortisone, or both, was inhibited by endotoxin. The incorporation of the (14)C-label from alanine-U-(14)C, pyruvate-2-(14)C, or glucose-U-(14)C into blood glucose and liver glycogen was substantially impaired in endotoxemic animals at 12 h. Decreases in incorporation occurred as early as 4 h after treatment. The progressive increase in glycogen synthase activity observed in fasted controls was not seen in endotoxin-poisoned mice. The administration of a glucose or pyruvate load to endotoxin-treated mice did not restore gluconeogenesis, glycogen synthesis, or liver glycogen synthase activity to normal levels. The in vivo activation of glycogen synthase by glucose was significantly reduced in endotoxemic animals. These changes indicate reduced carbohydrate synthesis as a probable cause for rapid sugar loss during endotoxemia in mice.

Red cell glycolysis in a case of 3-phosphoglycerate kinase deficiency.

Abstract. A severe deficiency in red cell 3‐phosphoglycerate kinase was observed in a 62‐year‐old woman with haemolytic anaemia. Compared with a normal “young” red cell population with the same degree of reticulocytosis (6–7%) the 3‐phosphoglycerate kinase activity was reduced to 27%. A concomitant decrease of hexokinase and pyruvate kinase (both reduced by about 30%) was observed. The activities of glucoses‐phosphate dehydrogenase, phosphofructokinase, fructose‐1,6‐di‐phosphate aldolase, gIyceraIdehyde‐3‐phosphate dehydrogenase and lactate dehydrogenase were slightly increased (7 to 15%). The total glycolytic output of the deficient cells was decreased by 28% at pH 7.0, by 36% at pH 7.4 and by 34% at pH 7.6. Compared with a normal “adult” red cell population the 3‐phosphoglycerate kinase activity was reduced to 42% of the control values. Hexokinase, glyceraldehyde‐3‐phosphate dehydrogenase and lactate dehydrogenase activities were increased by approximately 20–50%. Phosphofructokinase activity was unchanged and pyruvate kinase only slightly increased. The steady state levels of the intermediates preceding the 3‐phosphoglycerate kinase step were increased 2–3 fold. The subsequent metabolites were decreased or practically unmodified. ATP, ADP, NAD+, and NADH were not affected. The reduced glutathione level was increased and the ratio of reduced to oxidized glutathione was doubled. The glycolytic output and its pH‐dependency were normal. The metabolic significance of the enzyme defect was assessed by the in vitro creation of cell stressing conditions, i.e. low pH and high pyruvate levels. In both cases, the 3‐phosphoglycerate kinase activity became limiting at low pH, glucose‐6‐phosphate accumulated at a faster rate and fructose‐ 1,6‐diphosphate and dihydroxyacetone phosphate disappeared more slowly in the deficient cells. After pyruvate loading these cells showed: a faster, more pronounced rise in 1,3‐diphosphoglycerate and a decrease in 2,3‐diphosphoglycerate (slightly increased in the controls): a drop in reduced glutathione (constant in the controls): constant ATP and slightly increased 3‐phosphoglycerate concentrations (both strongly increased in the controls): a slight increase in NADH (dropped to nil in the controls). Steady state glycolysis under normal conditions seemed to be affected by the enzyme deficiency. 3‐phosphoglycerate kinase however, became more severely limiting a low pH or after the addition of pyruvate. In these conditions, the flow was diverted to the 2,3‐diphosphoglycerate bypass, less ATP was produced and the concentration of reduced glutathione decreased. This may be assumed to have led to impairment of the ionic pump and may thus explain the increased haemolysis.